Protein posttranslational modifications (PTMs) control much of biology. Glycosylation is one of the most common PTMs and more powerful tools are needed to study the occurrence and relative levels of glycosylation. In this proposal we describe the development of new multicolor fluorescent labeling reagents designed for powerful, differential glycomics analysis. The broad, long-term objectives of this work are to simultaneously monitor global changes in levels of (1) proteins, (2) a variety of post-translational modifications, and (3) enzyme activities--in response to biological variables, with greatly improved sensitivity. To accomplish these objectives we are developing (in a cooperative project between Zdye LLC and Montana State University) a new family of ultrasensitive, highly water soluble fluorescent dyes called Zdyes for multicolor, differential labeling--that do not shift the isoelectric points of Zdye-labeled proteins and tend to enhance recovery of labeled proteins. Most PTMs cause shifts in 2D gel spot positions and thus 2D gels provide global pictures of PTM patterns. Our recent work has focused on overcoming past limitations in 2D gel technology and measuring changes in protein levels with biological stimulation. Specific Aims: (1) We will use ortho hydroxymethyl-substituted arylboronic acids to form covalent boronate esters with 1,2-cis diols or 1,3-diols, which are present on all carbohydrates. The ortho hydroxymethyl substituent enhances the binding constants with glycopyranoses about one hundred fold. A benzophenone group will be attached to the arylboronic acid and irradiated to trap the reversible boronate complex. The irradiated benzophenone is designed to abstract a hydrogen atom from the carbohydrate backbone and form a carbon-carbon bond between the carbohydrate and the carbonyl carbon of the benzophenone. Excited benzophenone reacts very poorly with water and if it does not find a C-H to abstract it returns to the ground state to be excited again. Binding and photo-crosslinking will be characterized with a variety of mono and disaccharides. (2) Fluorescent Zdyes will be tethered to the enhanced phenyl boronic acid-benzophenone sugar linker and characterized for crosslinking to a variety of sugars. (3) Glycoprotein standards will be crosslinked with two different-colored Zdye Enhanced Sugar-linkers (Zdye ES-linkers) and conditions optimized for differential labeling and analysis with 1D and 2D gels. (4) Complex protein mixtures from mammalian cell lines expressing human glycoproteins and rat brain samples will be labeled with different colored Zdye ES-linkers and Zdye protein linkers and analyzed on 2D gels to quantify the relative amounts of glycosylation under different biological conditions. Dr. Edward Dratz, PI, is an expert in proteomics, Dr. Paul Grieco, co-PI, is a leader in total organic synthesis, Dr. Mary Cloninger, co-PI, is experienced in carbohydrates and glycoproteins, and Dr. Don Thorne, CEO Zdye LLC has a PhD in pharmacology and expertise in glycoprotein research and business development: a highly synergistic team to develop new glycomics tools. Glycoproteins serve crucial roles in cellular communication and mediate many mechanisms in health and disease. For example, carbohydrate post-translational protein modifications are central to viral and bacterial infection, for metastatic spread of cancer cells, for controlling cellular differentiation, for mounting immune responses, and are involved in modulating transcription factor activity - that control gene expression, in an analogous manner to reversible phosphorylation. Tools to study glycoproteins, however, lag in development at this time and we describe the development of new fluorescence detection technology to determine the relative amounts of glycoproteins in samples exposed to different biological variables, which promise significant advantages for biomedical research.